The production of proteins for biopharmaceutical applications typically involves the use of cell cultures that are known to produce proteins exhibiting varying levels of heterogeneity. The basis for such heterogeneity includes, but is not limited to, the presence of distinct glycosylation substitution patterns. For example, such heterogeneity can be observed in increases in the fraction of proteins substituted with agalactosyl fucosylated biantennary oligosaccharides NGA2F+NGA2F-GlcNAc and decreases in the fraction of proteins substituted with galactose-containing fucosylated biantennary oligosaccharides NA1F+NA2F. Such heterogeneity can be assayed by releasing oligosaccharides present on the protein of interest via enzymatic digestion with N-glycanase. Once the glycans are released, the free reducing end of each glycan can be labeled by reductive amination with a fluorescent tag. The resulting labeled glycans are separated by normal-phase HPLC(NP-HPLC) and detected by a fluorescence detector for quantitation.
Technological advances in recombinant protein production analysis have provided unique opportunities for identifying the extent of heterogeneity exhibited by a particular protein population, particularly in the context of large-scale production of recombinant proteins. Although such advances have allowed for the robust characterization of protein heterogeneity, there remains a need in the art to identify culture conditions and production methods that allow for control over the development of such heterogeneity. Control of protein heterogeneity is particularly advantageous in the context of cell culture processes used for commercially produced recombinant bio-therapeutics as such heterogeneity has the potential to impact therapeutic utility. The instant invention addresses this need by providing compositions and processes to control protein heterogeneity.